Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Vinblastine-isolated microtubule protein from chick embryonic muscles has an enzymatic activity which catalyzes the formation of phosphatidic acid from diglycerides and ATP. The pH optimum (6.4), sedimentation on sucrose gradients (Mr = 85 000), and sensitivity to ions of this diglyceride kinase activity are different to those of a similar enzymatic activity present in 150 000 X g supernatants of chick embryonic muscle homogenates, suggesting that it is a different species which is associated specifically with the microtubules. The reaction requires a divalent ion (e.g. 0.4 mM Mg2+ gives half-maximal stimulation), and GTP can replace ATP rather effectively, especially at nucleotide concentrations lower than 50 muM. The sedimentation of the diglyceride kinase on sucrose gradients coincides with that of the microtubules-associated protein kinase (Mr = 75 000); the heat-stability and sensivitity to proteolysis of both activities are also very similar. Stimulation of one reaction by the addition of the corresponding exogenous substrate does not impair the phosphorylation of the other, and no radioactivity is lost from phosphatidic acid or the protein moiety upon incubation of pre-labelled microtubules with a large excess of unlabelled ATP or GTP. In addition to diglyceride and protein kinase activities (0.2 and 0.3 nmol 32P-transferred X min-1 X mg-1 microtubular protein, respectively), microtubules also contain an associated ATPase (2.8 nmol X min-1 X mg-1), which requires either Mg2+ or Ca2+, can hydrolyze GTP quite effectively, and sediments with a molecular weight of 95000. The results obtained are discussed in connection with the possible relationships existing among these enzymatic activities, as well as their probable role in microtubular functions.
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PMID:Diglyceride kinase activity of microtubules. Characterization and comparison with the protein kinase and ATPase activities associated with vinblastine-isolated tubulin of chick embryonic muscles. 18 51

Esters of succinic acid stimulate insulin secretion from pancreatic beta-cells. Using collagenase-isolated rat islets, the transduction mechanisms involved were investigated. In freshly isolated perifused islets, monomethyl succinate (MMSucc), in the presence of basal (2.75 mM) glucose, stimulated insulin release in a biphasic pattern. This secretory response was dependent on extracellular calcium movement into the beta-cell, since the calcium channel blocker nitrendipine (5 microM) abolished it. The glucokinase inhibitor mannoheptulose (20 mM) had no effect on its secretory action, while the protein kinase-C inhibitor staurosporine (20 nM) reduced secretion to MMSucc. In addition, while ineffective alone, the diacylglycerol kinase inhibitor monooleoylglycerol (25 microM) potentiated MMSucc-induced insulin release. A similarly amplified response occurred in the presence of forskolin (0.25 microM), a compound that elevates islet cAMP levels. The sodium salt of succinic acid (20 mM) had no effect on insulin release in the presence or absence of forskolin. Prior treatment with MMSucc in the presence of 2.75 mM glucose sensitized islets to the usually weak insulin secretory effect of 7.5 mM glucose. Other groups of islets were incubated for 2 h with myo-[2-3H]inositol to label their phosphoinositide pools. These islets were subsequently stimulated, and the kinetics of [3H]inositol efflux and insulin secretion were measured. MMSucc induced a rapid and sustained dose-dependent increase in [3H]inositol efflux rates. In batch-incubated islets, MMSucc increased inositol phosphate levels. Finally, MMSucc (20 mM), in the presence of 8 mM glucose, did not influence the detritiation of [5-3H]glucose, but reduced the oxidation of [U-14C] glucose. These results support the following conclusions. First, MMSucc is a potent activator of islet phosphoinositide hydrolysis. Second, the activation of protein kinase-C appears to contribute to the acute insulin secretory effect of MMSucc. Third, MMSucc-induced increases in phosphoinositide hydrolysis contribute at least in part to its ability to acutely stimulate insulin release and prime the beta-cell to subsequent stimulation. Finally, mitochondrial events associated with the oxidative metabolism of MMSucc may underlie its insulinotropic action.
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PMID:Biochemical mechanisms involved in monomethyl succinate-induced insulin secretion. 132 78

We reported previously that in pancreatic islet cells, certain diacylglycerols (DGs) evoke increases in cytosolic calcium ([Ca2+]i), mainly by intracellular mobilization. We now examined the effects of DGs on the increase in [Ca2+]i due to Ca2+ influx. In the insulin-secreting HIT T-15 islet cell line, cell membrane depolarization using 40 mM KCl evoked a 2- to 3-fold increase in [Ca2+]i, which lasted several minutes. A cell-permeable DG, 1,2-dioctanoylglycerol (DiC8; 10 microM) induced a 12 +/- 4% rise in [Ca2+]i, which did not occur in the absence of extracellular Ca2+ or in the presence of verapamil; this effect was not protein kinase-C (PKC) dependent, because it was not altered by the addition of the PKC inhibitor staurosporine or by using PKC-depleted cells. When DiC8 was added first, the KCl-induced increase in [Ca2+]i was inhibited in a dose-dependent manner (100% at 10-15 microM DiC8); this effect was PKC independent. At a concentration of 10 microM, other synthetic DGs, 1,2-dihexanoylglycerol (DiC6), 1,2-didecanoylglycerol (DiC10), or 1-oleoyl-2-acetylglycerol, inhibited the KCl-induced rise in [Ca2+]i to 15 +/- 4%, 47 +/- 7%, and 51 +/- 5% of the control value, respectively. R59022 (10 microM), which inhibits DG kinase and causes accumulation of endogenous DGs, inhibited the KCl-induced rise in [Ca2+]i to 2 +/- 0.2% of the control value; this inhibition was not affected by staurosporine. In anchored cells, KCl stimulated insulin release (959 +/- 88 microU/mg protein above the control value); 20 microM DiC6 or DiC8 attenuated KCl-induced insulin release by 68% and 31% of the control value, respectively; DiC10 or 1-oleoyl-2-acetylglycerol had no effect. R59022 inhibited KCl-induced insulin release by 90% of the control value. We conclude that in HIT T-15 cells, DGs may serve as positive and negative modulators of [Ca2+]i, apparently by complex and PKC-independent mechanisms. These divergent actions of DGs on islet cell Ca2+ balance together with the accompanying activation of PKC affect insulin release in a complex manner.
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PMID:Diacylglycerol inhibits potassium-induced calcium influx and insulin release by a protein kinase-C-independent mechanism in HIT T-15 islet cells. 139 42

In the first report in this series we described the relationships and evolution of 152 individual proteins of the EF-hand subfamilies. Here we add 66 additional proteins and define eight (CDC, TPNV, CLNB, LPS, DGK, 1F8, VIS, TCBP) new subfamilies and seven (CAL, SQUD, CDPK, EFH5, TPP, LAV, CRGP) new unique proteins, which we assume represent new subfamilies. The main focus of this study is the classification of individual EF-hand domains. Five subfamilies--calmodulin, troponin C, essential light chain, regulatory light chain, CDC31/caltractin--and three uniques--call, squidulin, and calcium-dependent protein kinase--are congruent in that all evolved from a common four-domain precursor. In contrast calpain and sarcoplasmic calcium-binding protein (SARC) each evolved from its own one-domain precursor. The remaining 19 subfamilies and uniques appear to have evolved by translocation and splicing of genes encoding the EF-hand domains that were precursors to the congruent eight and to calpain and to SARC. The rates of evolution of the EF-hand domains are slower following formation of the subfamilies and establishment of their functions. Subfamilies are not readily classified by patterns of calcium coordination, interdomain linker stability, and glycine and proline distribution. There are many homoplasies indicating that similar variants of the EF-hand evolved by independent pathways.
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PMID:Evolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories. 160 95

Diacylglycerol (DG) and its analogue phorbol 12-myristate 13-acetate (PMA) activate the ubiquitous phospholipid/Ca2(+)-dependent protein kinase, protein kinase C (PKC), and cause it to become tightly associated with membranes. DG is produced transiently as it is rapidly metabolized by DG kinase (DGK) to phosphatidic acid. Phorbol esters such as PMA are not metabolized and induced a prolonged membrane association of PKC. Until recently, PKC was the only known phorbol ester receptor. We have shown that a novel brain-specific cDNA, neuronal chimaerin (NC), expressed in Escherichia coli, binds phorbol ester with high affinity, stereospecificity and a phospholipid requirement [Ahmed, Kozma, Monfries, Hall, Lim, Smith & Lim (1990) Biochem. J. 272, 767-773]. The proteins NC, PKC and DGK possess a cysteine-rich domain with the motif HX11/12CX2CXnCX2CX4HX2CX6/7C (where n varies between 12 and 14). The partial motif, CX2CX13CX2C, is present in a number of transcription factors including the steroid hormone receptors and the yeast protein, GAL4, in which zinc plays a structural role of co-ordinating cysteine residues and is essential for DNA binding (protein-nucleic acid interactions). The cysteine-rich domain of NC and PKC is required for phospholipid-dependent phorbol is required for phospholipid-dependent phorbol ester binding, suggesting an involvement of this domain in protein-lipid interactions. We have expressed recombinant NC, PKC and DGK glutathione S-transferase and TrpE fusion proteins in E. coli to investigate the relationship between the cysteine-rich motif, HX11/12CX2CX10-14CX2CX4HX2CX6/7C, zinc and phorbol ester binding. The cysteine-rich domain of NC, PKC and DGK bound 65Zn2+ but only NC and PKC bound [3H]phorbol 12,13-dibutyrate. When NC and PKC were subjected to treatments known to remove metal ions from GAL4 and the human glucocorticoid receptor, phorbol ester binding was inhibited. These data provide evidence for the role of a zinc-dependent structure in phorbol ester binding.
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PMID:The cysteine-rich domain of human proteins, neuronal chimaerin, protein kinase C and diacylglycerol kinase binds zinc. Evidence for the involvement of a zinc-dependent structure in phorbol ester binding. 166 Feb 66

Cytosolic diacylglycerol kinase was irreversibly inactivated by 5'-AMP since the enzyme remained less active after the removal of 5'-AMP by P-10 gel chromatography. The inactivation was time-dependent, suggesting the involvement of a covalent bond modification. A reconstitution experiment detected a rat brain cytosolic mediator for the effect of 5'-AMP. A protein kinase rich fraction prepared from rat liver was also capable of restoring the sensitivity of diacylglycerol kinase-II to 5'-AMP. We propose that 5'-AMP-activated protein kinase is the mediator which inactivates diacylglycerol kinase-II, possibly by phosphorylation.
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PMID:Irreversible inactivation of diacylglycerol kinase-II requires a mediator. 166 22

NIH 3T3 fibroblasts were transfected with the chloramphenicol-acetyltransferase (CAT) gene under the control of the SV40 early promoter, which can be stimulated by IL-1. CAT activity in cell lysates and PGE2 release in the supernatants were measured in control and stimulated cell cultures in parallel. Human IL-1 beta (180 pM) and human rTNF-alpha (3 nM) significantly stimulated both CAT activity and PGE2 release. The combined incubation of the two cytokines resulted in a synergistic effect on PGE2 release. The addition of AA (30 microM) greatly stimulated PGE2 release without affecting CAT activity. Similarly, drugs interfering with AA metabolism were without effect on CAT activity although profoundly reducing PGE2 release. Forskolin (0.1 microM) did not modify either parameter. The glucocorticoid fluocinolone (20 nM) was able to decrease both parameters. Protein kinase inhibitors H7 (5-50 microM) and sphingosine (50 microM) inhibited only IL-1-induced CAT activity, whereas H8 (5-50 microM) and HA1004 (50 microM) were ineffective on both parameters. PMA (0.5 microM) and R59 022, a diacylglycerol kinase inhibitor (10 microM), did not modify either control or IL-1-induced CAT activity. IL-1-stimulated PGE2 release was potentiated by PMA, although this effect was not inhibited by H7. The data suggest that: 1) in NIH 3T3 cells the activation of AA metabolism by IL-1 is not involved in IL-1-induced gene expression; 2) protein kinase C activity is required but not sufficient for IL-1-induced gene expression; and 3) PMA may stimulate AA metabolism by a mechanism in part independent of protein kinase activity.
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PMID:Induction of gene expression by IL-1 in NIH 3T3 cells. Possible requirement of protein kinase C activity and independence from arachidonic acid metabolism. 212 35

Phosphatidylinositol 4-phosphate (PIP) kinase (E.C. 2.7.1.68) has been purified about 1200-fold from rat liver plasma membranes, taking advantage of affinity chromatography on quercetin-Sepharose as a novel step. The purified PIP kinase showed no contamination by the following enzyme activities: phosphatidylinositol (PI) kinase (EC 2.7.1.67), protein kinase C (EC 2.7.1.-), diacylglycerol kinase (EC 2.7.1.-), phospholipase C (EC 3.1.4.11), protein-tyrosine kinase (EC 2.7.1.112), alkaline phosphatase (EC 3.1.3.1), triphosphoinositide phosphomonoesterase (EC 3.1.3.36), adenylate kinase (EC 2.7.4.3) and cAMP-dependent protein kinase (EC 2.7.1.37). The liver membrane enzyme requires high Mg2+ concentrations with a KM value of 10 mM. Ca2+ or Mn2+ could replace Mg2+ to a certain, though small, extent. Apparent KM values with respect to PIP and ATP were 10 and 65 microM, respectively. GTP was slightly utilized by the kinase as phosphate donor while CTP was not. Quercetin inhibited the enzyme with Ki = 34 microM. Extending our previous observations (Urumow, T. and Wieland, O.H. (1986) FEBS Lett. 207, 253-257 and Urumow, T. and Wieland, O.H. (1988) Biochim. Biophys. Acta 972, 232-238) [gamma S]pppG still stimulated the PIP kinase in extracts of solubilized liver membranes. 20-40% (NH4)2SO4 precipitation of the membrane extracts yielded a fraction that contained the bulk of enzyme activity but did not respond to stimulation by [gamma S]pppG any longer. This was restored by recombination with a protein fraction collected at 40-70% (NH4)2SO4 saturation, presumably containing a GTP binding protein and/or some other factor separated from the PIP kinase. In the reconstituted system [gamma S]pppG stimulated PIP kinase in a concentration dependent manner with maximal activation at 5 microM. This effect was not mimicked by [gamma S]pppA and was blocked by [beta S]ppG. These results strongly support our view that in liver membranes PIP kinase is regulated by a G-protein.
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PMID:Purification and partial characterization of phosphatidylinositol-4-phosphate kinase from rat liver plasma membranes. Further evidence for a stimulatory G-protein. 215 97

We investigated the effects of enzyme phosphorylation in vitro on the properties of diacylglycerol kinase. Diacylglycerol kinase and protein kinase C, both present as Mr-80,000 proteins, were highly purified from pig thymus cytosol. Protein kinase C phosphorylated diacylglycerol kinase (up to 1 mol of 32P/mol of enzyme) much more actively than did cyclic AMP-dependent protein kinase. Phosphorylated and non-phosphorylated diacylglycerol kinase showed a similar pI, approx. 6.8. Diacylglycerol kinase phosphorylated by either protein kinase C or cyclic AMP-dependent protein kinase was almost exclusively associated with phosphatidylserine membranes. In contrast, soluble kinase consisted of the non-phosphorylated form. The catalytic properties of the lipid kinase were not much affected by phosphorylation, although phosphorylation-linked binding with phosphatidylserine vesicles resulted in stabilization of the enzyme activity.
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PMID:Phosphorylation of diacylglycerol kinase in vitro by protein kinase C. 253 7

In isolated guinea pig parotid gland lobules the activities of the following enzymes were measured 30 sec after stimulation with either 2 X 10(-5) M isoproterenol or 10(-5) M carbachol: glycerol kinase (EC 2.7.1.30), glycerolphosphate acyltransferase (EC 2.3.1.15), lysophosphatidate acyltransferase (EC 2.3.1.51), phosphatidate phosphohydrolase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol kinase (EC 2.7.1.107), and CDP-diacylglycerol synthetase (EC 2.7.7.41). Lyso-phosphatidate acyltransferase, diacylglycerol kinase, and diacylglycerol acyltransferase exhibited significant increases following stimulation by both types of agonists. Stimulation of the activities of these three enzymes occurred also following in vitro incubation with the catalytic subunit of cAMP-dependent protein kinase or a Ca2+/calmodulin-dependent protein kinase II. These effects could be reversed by incubation with various protein phosphatases. When cells were first stimulated with either type of agonist, subsequent incubation with protein kinases was almost ineffective. Activation by the two types of protein kinases was not additive, indicating that they activate by phosphorylating identical sites on the enzyme proteins. The other enzymes examined showed no or only minor changes and their activities could not be affected by in vitro incubation with the two types of protein kinases. The results explain the rapid changes in acyl-group transfer from acyl-CoA to neutral lipids observed previously during the first seconds after stimulation of guinea pig parotid gland lobules with isoproterenol or carbachol (1). An analysis of a potential role of lipocortins for the regulation of phosphoinositide-specific phospholipases C reveals that these proteins do indeed inhibit these enzymes, but that this inhibition results from a calcium-dependent interaction of the lipocortins with the phospholipid substrate. A physiological role of lipocortins for the regulation of phospholipases is doubtful.
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PMID:Mechanisms of short-term (second range) regulation of the activities of enzymes of lipid and phospholipid metabolism in secretory cells. 256 Mar 28


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